The Role of Rac1 in the Growth Cone Dynamics and Force Generation of DRG Neurons

We used optical tweezers, video imaging, immunocytochemistry and a variety of inhibitors to analyze the role of Rac1 in the motility and force generation of lamellipodia and filopodia from developing growth cones of isolated Dorsal Root Ganglia neurons. When the activity of Rac1 was inhibited by the drug EHop-016, the period of lamellipodia protrusion/retraction cycles increased and the lamellipodia retrograde flow rate decreased; moreover, the axial force exerted by lamellipodia was reduced dramatically. Inhibition of Arp2/3 by a moderate amount of the drug CK-548 caused a transient retraction of lamellipodia followed by a complete recovery of their usual motility. This recovery was abolished by the concomitant inhibition of Rac1. The filopodia length increased upon inhibition of both Rac1 and Arp2/3, but the speed of filopodia protrusion increased when Rac1 was inhibited and decreased instead when Arp2/3 was inhibited. These results suggest that Rac1 acts as a switch that activates upon inhibition of Arp2/3. Rac1 also controls the filopodia dynamics necessary to explore the environment

Characterization of lamellipodial protrusion/retraction cycles and of vertical motion.

<p><b>(a-c)</b> From top to bottom: images of the lamellipodium undergoing cyclic waves of protrusion (t₂) and retraction (t₁ and t₃) in control conditions; the white dotted line represents the leading edge of the lamellipodium_. Scale bar, 5 μm. <b>(d)</b> The profile diagram of the positions of the lamellipodium edge during the time course. Increase in the color intensity shows increase in the frequency of the lamellipodia edge to be present at particular location. White lines used to plot the kymographs. <b>(e)</b> Kymograph showing the protrusion/retraction cycles of lamellipodia. White dots show the leading edge of lamellipodia. The characteristic values of period of protrusion/retraction cycles of lamellipodia motion (black dotted line), the retrograde flow rate (black line) and persistence length of lamellipodia (white line) i.e. (T), (dx/dt) and (dl) respectively were calculated along the label lines. <b>(f)</b> Fractional number of pixels in focus at 5μm height above the coverslip. The protrusion/retraction cycles of lamellipodia is also observed in terms of fractional reached height by lamellipodia. The black line shows the peak position of the fractional height where lamellipodia reach the maximum in axial direction at the end of the retraction.</p

The feedback and nanopositioner system.

<p>(a-c) High-resolution images of a bead trapped in front of a lamellipodium emerging from the soma of a DRG neuron in control conditions and during a push. At t₁ the bead is in the optical trap (a) Scale bar, 2μm.The lamellipodium grows, at t₂, tries to push the bead out of the trap (b). At t₃ the feedback mechanism of the Nano-drive redirects the bead back into the centre of the trap (c). The red cross indicates the center of the optical trap. (d) The X, Y components of the trace. The position of the bead (blue and green curve respectively, upper panel), compensated the X, Y position of the bead (magenta and light green curve, upper panel), corresponding to X, Y position of the Nanodrive (Blue and green respectively, lower panel).</p

Rac1 restores lamellipodia’s motion after transient retraction when Arp2/3 is inhibited.

<p><b>(a)</b> Kymograph (upper panel) and fractional height reached by lamellipodia (lower panel) in control conditions (before the black vertical line) and in the presence of 100 μM CK (after the vertical black line). <b>(b)</b> As in (a) but in the presence of 50 μM CK (green line) and of 20 μM EH (blue line). <b>(c)</b> As in (a) but in the presence of 50 μM CK and of 20 μM EH together (yellow vertical line). <b>(d)</b> As in (a) but in the presence of 50 μM ZCL (purple vertical line) and of 50 μM CK (green vertical line). <b>(e)</b> As in (a) but in the presence of 1μg/ml CT04 (brown vertical line) and 50 μM CK (green vertical line). Vertical lines show time at which the inhibitors were added. We observed the same behavior in each case for n ≥ 8 experiments. <b>(f)</b> Period of protrusion/Retraction cycles of lamellipodia in control conditions, with 50 μM ZCL (13 min), with 1μg/ml CT (13 min) and 500nm GSK (13 min). <b>(g)</b> Persistence length of lamellipodia in control conditions, with 50 μM ZCL (13 min), with 1μg/ml CT (13 min) and 500nm GSK (13 min). <b>(h)</b> Retrograde flow rate of lamellipodia in control conditions, with 50 μM ZCL (13 min), with 1μg/ml CT (13 min) and 500nm GSK (13 min). Student t-test showed that the data significantly differ from the control conditions, *P<0.05. Data represent mean ± SEM. <b>(i)</b> Quantification of Rac1-GTP level in DRG neurons in control, 25 μM CK (after 2 min), 50 μM CK (after 2 min) and 50 μM CK (after 8 min) conditions. Student t-test showed that the data significantly differ from the control conditions, n = 8,**P<0.005. Data represent mean ± SEM.</p

The effect of the CK and EH on the motility and force exerted by filopodia.

<p><b>(a-b)</b> Phase contrast images of GC before and after treatment with 20 μM EH. Note the length of filopodia in each case. Scale bar 5 μm. <b>(c-d)</b> Staining of F-actin by phalloidin in GC before and after treatment with 20 μM EH. <b>(e)</b> Rate of filopodia protrusion in control conditions (red), with 25 μM CK (green), with 50 μM CK (dark green), with 10 μM EH (cyan) and with 20 μM EH (blue). <b>(f)</b> Maximum length of filopodia in control conditions (red), with 25 μM CK(green), with 50 μM CK(dark green), with 10 μM EH (cyan) and with 20 μM EH (blue). <b>(g)</b> Images of a bead trapped in front of a filopodium emerging from a GC of DRG neuron in the presence of 25 μM CK. At t₁ the bead is in the optical trap and at t₂ the filopodium pushes the bead. The cross indicates the centre of the optical trap. (<b>h)</b> The three components F_x, F_y and F_z of the force exerted by the filopodium in the presence of 25 μM CK. <b>(i-k)</b> As in (h) but in the presence of 50 μM CK (i), in the presence of 10 μM EH (j) and in the presence of 20 μM EH (k) respectively. <b>(l)</b> Filopodia force in control conditions (red), in the presence of 25 μM CK (green), of 50 μM CK (dark green), of 10 μM EH (cyne) and of 20 μM EH (blue). The trap stiffness was k_x,y = 0.10 pN/nm, k_z = 0.08 pN/nm. By using the student t-test, the data differs with respect to the control conditions with a significance of *P<0.05 and **P<0.005. Data represent mean ± SEM. All the data were checked with chi-square test for Normal distribution before applying the student’s t test.</p

Cancer Epidemiology and Control in the Arab World - Past, Present and Future

The Arab world, stretching from Lebanon and Syria in the north, through to Morocco in the west, Yemen in the south and Iraq in the east, is the home of more than 300 million people. Cancer is already a major problem and the lifestyle changes underlying the markedly increasing rates for diabetes suggest that the burden of neoplasia will only become heavier over time, especially with increasing obesity and aging of what are now still youthful populations. The age-distributions of the affected patients in fact might also indicate cohort effects in many cases. There are a number of active registries in the region and population-based data are now available for a considerable number of countries. A body of Arab scientists are also contributing to epidemiological research into the causes of cancer and how to develop effective control programs. The present review covers the relevant PubMed literature and cancer incidence data from various sources, highlighting similarities and variation in the different cancer types, with attempts to explain disparities with reference to possible environmental factors. In males, the predominant cancers vary, with lung, urinary bladder or liver in first place, while for females throughout the region breast cancer is the greatest problem. In both sexes, non-Hodgkins lymphomas and leukemias are relatively frequent, along with thyroid cancer in certain female populations. Adenocarcinomas of the breast, prostate and colorectum appear to be increasing. Coordination of activities within the Arab world could bring major benefits to cancer control in the eastern Mediterranean region